A Mg2+-dependent RNA Tertiary Structure Forms in the Bacillus subtilis trp Operon Leader Transcript and Appears to Interfere with trpE Translation Control by Inhibiting TRAP Binding

Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti- trp RNA-binding attenuation protein

“…3) revealed a mean T 1 for AT of 1.91 AE 0.25 s, whereas that in fAT is 0.631 AE 0.091 s. The mean 15 N T 2 of AT is 31.9 AE 4.5 ms, whereas that of fAT is 85 AE 13 ms. Fitting these data to an isotropic tumbling model (32) yielded overall rotational correlation times of 25.2 AE 2.8 ns for AT and 8.2 AE 1.0 ns for fAT. Hydrodynamic calculations (36) using coordinates derived from the AT 12 crystal structure (21) predicted an isotropic rotational correlation time at 55°C of τ m ¼ 28.7 ns for AT 12 , and an axially symmetric rotational diffusion tensor for AT 3 with correlations times of τ zz m ¼ 8.4 ns and τ ⊥ m ¼ 6.3 ns. Hydrodynamic properties were also measured in one-dimensional stimulated echo gradient diffusion NMR experiments (37), which yielded diffusion coefficients of D AT 298 ¼ 5.68 × 10 −7 cm 2 ∕s and D fAT 298 ¼ 9.56 × 10 −7 cm 2 ∕s for AT and fAT, respectively (Fig.…”

“…Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT 12 , composed of a tetramer of trimers, AT 3 . Structural and biochemical studies suggest that only trimeric AT is active for binding to TRAP.…”

“…Hydrodynamic parameters from NMR relaxation and diffusion measurements showed that fAT is exclusively trimeric (AT 3 ), while (deformylated) AT exhibits slow exchange between both trimeric and dodecameric forms. We examined this equilibrium using NMR spectroscopy and found that oligomerization of active AT 3 to form inactive AT 12 is linked to protonation of the amino terminus. Global analysis of the pH dependence of the trimer-dodecamer equilibrium revealed a near physiological pK a for the N-terminal amine of AT and yielded a pH-dependent oligomerization equilibrium constant.…”

“…In the absence of cellular tryptophan, the antiterminator structure is favored, allowing transcription and translation of the operon. When cellular tryptophan levels are high, the amino acid binds to TRAP, which becomes activated for binding to an RNA site that overlaps the antiterminator sequence, favoring formation of the terminator; translation of these genes is regulated by a similar mechanism (1,6,9,10,(12)(13)(14). TRAP is a small protein (∼74 amino acids) that assembles into an undecameric (11-mer) ring-shaped structure, with binding sites for tryptophan arranged between neighboring TRAP protomers (15,16).…”

Translation Control of trpG from Transcripts Originating from the Folate Operon Promoter of Bacillus subtilis Is Influenced by Translation-Mediated Displacement of Bound TRAP, While Translation Control of Transcripts Originating from a Newly Identified trpG Promoter Is Not

“…Filter binding reactions were performed following a published procedure (24). Purified TRAP and labeled RNAs were identical to those described for the gel mobility shift assay.…”